1. Field of the Invention
The present invention is in the field of recombinant genetics, protein expression, and vaccines. The present invention relates, in particular, to a method of expressing in a recombinant host an outer membrane group B porin protein from Neisseria meningitidis. The invention also relates to a method of purification and refolding of the recombinant protein.
2.Background Information
The outer membranes of Neisseria species much like other Gram negative bacteria are semi-permeable membranes which allow free flow access annd escape of small molecular weight substances to and from the periplasmic space of these bacteria but retard molecules of larger size (Heasley, F. A., et al., "Reconstitution and characterization of the N. gonorrhoeae outer membrane permeability barrier," in Genetics and Immunobiology of Neisseria gonorrhoeae, Danielsson and Nomark, eds., University of Umea, Umea, pp. 12-15 (1980); Douglas, J. T., et at., FEMS Microbiol. Lett. 12:305-309 (1981)). One of the mechanisms whereby this is accomplished is the inclusion within these membranes of proteins which have been collectively named porins. These proteins are made up of three identical polypeptide chains (Jones, R. B., et at., Infect. Immun. 30:773-780 (1980); McDade, Jr. and Johnston, J. Bacteriol. 141:1183-1191 (1980)) and in their native trimer conformation, form water filled, voltage-dependent channels within the outer membrane of the bacteria or other membranes to which they have been introduced (Lynch, E. C., et al., Biophys. J. 41:62 (1983); Lynch, E. C., et al., Biophys. J. 45:104-107 (1984); Young, J. D. E., et at., Proc. Natl. Acad. Sci. USA 80:3831-3835 (1983); Mauro, A., et al., Proc. Natl. Acad. Sci. USA 85:1071-1075 (1988); Young, J. D., et al., Proc. Natl. Acad. Sci. USA 83:150-154 (1986)). Because of the relative abundance of these proteins within the outer membrane, these protein antigens have also been used to subgroup both Neisseria gonorrhoeae and Neisseria meningitidis into several serotypes for epidemiological purposes (Frasch, C. E., et al., Rev. Infect. Dis. 7:504-510 (1985); Knapp, J. S., et al., "Overview of epidemiological and clinical applications of auxotype/serovar classification of Neisseria gonorrhoeae," The Pathogenic Neisseriae, Schoolnik, G. K., ed., American Society for Microbiology, Washington, pp. 6-12 (1985)). To date, many of these proteins from both gonococci and meningococci have been purified (Heckels, J. E., J. Gen. Microbiol. 99:333-341 (1977); James and Heckels, J. Immunol. Meth. 42:223-228 (1981); Judd, R. C., Anal. Biochem. 173:307-316 (1988); Blake and Gotschlich, Infect. Immun. 36:277-283 (1982); Wetzler, L. M., et al., J. Exp. Med. 168:1883-1897 (1988)), and cloned and sequenced (Gotschlich, E. C., et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); McGuinness, B., et al., J. Exp. Med. 171:1871-1882 (1990); Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987); Feavers, I. M., el at., Infect. Immun. 60:3620-3629 (1992); Murakami, K., et al., Infect. Immun. 57:2318-2323 (1989); Wolff and Stem, FEMS Microbiol. Lett. 83:179-186 (1991); Ward, M. J., et al., FEMS Microbiol. Lett. 73:283-289 (1992)).
The porin proteins were initially co-isolated with lipopolysaccharides. Consequently, the porin proteins have been termed "endotoxin-associated proteins" (Bjornson et al., Infect. Immun. 56:1602-1607 (1988)). Studies on the wild type porins have reported that full assembly and oligomerization are not achieved unless LPS from the corresponding bacterial strain is present in the protein environment (Holzenburg et al., Biochemistry 28:4187-4193 (1989); Sen and Nikaido, J. Biol. Chem. 266:11295-11300 (1991)).
The meningococcal porins have been subdivided into three major classifications which in antedated nomenclature were known as Class 1, 2, and 3 (Frasch, C. E., et al., Rev. Infect. Dis. 7:504-510 (1985)). Each meningococcus examined has contained one of the alleles for either a Class 2 porin gene or a Class 3 porin gene but not both (Feavers, I. M., et al., Infect. Immun. 60:3620-3629 (1992)); Murakami, K., et al., Infect. Immun. 57:2318-2323 (1989)). The presence or absence of the Class 1 gene appears to be optional. Likewise, all probed gonococci contain only one porin gene with similarities to either the Class 2 or Class 3 allele (Gotschlich, E. C., et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987)). N. gonorrhoeae appear to completely lack the Class 1 allele. The data from the genes that have been thus far sequenced would suggest that all neisserial porin proteins have at least 70% homology with each other with some variations on a basic theme (Feavers, I. M., et al., Infect. Immun. 60:3620-3629 (1992)). It has been suggested that much of the variation seen between these neisserial porin proteins is due to the immunological pressures brought about by the invasion of these pathogenic organisms into their natural host, man. However, very little is known about how the changes in the porin protein sequence effect the functional activity of these proteins.
It has been previously reported that isolated gonococcal porins of the Class 2 allelic type behave electrophysically somewhat differently than isolated gonococcal porins of the Class 3 type in lipid bilayer studies both in regards to their ion selectivity and voltage-dependence (Lynch, E. C., et al., Biophys. J. 41:62 (1983); Lynch, E. C., et al., Biophys. J. 45:104-107 (1984)). Furthermore, the ability of the different porins to enter these lipid bilayers from intact living bacteria seems to correlate not only with the porin type but also with the neisserial species from which they were donated (Lynch, E. C., et al., Biophys. J. 45:104-107 (1984)). It would seem that at least some of these functional attributes could be related to different areas within the protein sequence of the porin. One such functional area, previously identified within all gonococcal Class 2-like proteins, is the site of chymotrypsin cleavage. Upon chymotrypsin digestion, this class of porins lack the ability to respond to a voltage potential and close. Gonococcal Class 3-like porins as well as meningococcal porins lack this sequence and are thus not subject to chymotrypsin cleavage but nonetheless respond by closing to an applied voltage potential (Greco, F., "The formation of channels in lipid bilayers by gonococcal major outer membrane protein," thesis, The Rockefeller University, New York (1981); Greco, F., et al., Fed. Proc. 39:1813 (1980)).
The major impediment for such studies has been the ability to easily manipulate the porin genes by modem molecular techniques and obtain sufficient purified protein to carry out the biophysical characterizations of these altered porin proteins. It was early recognized that cloned neisserial porin genes, when expressed in Escherichia coli, were lethal to the host E. coli (Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987); Carbonetti, N. H., et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988); Barlow, A. K., et al., Infect. Immun. 55:2734-2740 (1987)). Thus, many of these genes were cloned and sequenced as pieces of the whole gene or placed into low copy number plasmids under tight expression control (Carbonetti, N. H., et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)). Under these conditions, even when the entire porin gene was expressed, very little protein accumulated that could be further purified and processed for characterization.
Another tack to this problem which has met with a modicum of success has been to clone the porin genes into a low copy, tightly controlled expression plasmid, introduce modifications to the porin gene, and then reintroduce the modified sequence back into Neisseria (Carbonetti, N. H., et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)). However, this has also been fraught with problems due to the elaborate restriction endonuclease system present in Neisseria, especially gonococci (Davies, J. K., Clin. Microbiol. Rev. 2:S78-S82 (1989)).
The present invention is directed to an approach to overcome these difficulties. The DNA sequence of the mature porin proteins, e.g. class 2 and class 3 as well as fusions thereof, my be amplified using the chromosome of the meningococcal bacteria as a template for the PCR reaction. The amplified porin sequences were ligated and cloned into an expression vector containing the T7 promoter. E. coli strain BL21 lysogenic for the DE3 lambda phage (Studier and Moffatt, J. Mol. Biol. 189:113-130 (1986)), modified to eliminate the ompA gene, was selected as one expression host for the pET-17b plasmid containing the porin gene. Upon induction, large amounts of the meningococcal porin proteins accumulated within the E. coli without any obvious lethal effects to the host bacterium. The expressed meningococcal porin proteins were extracted and processed through standard procedures and finally purified by molecular sieve chromatography and ion exchange chromatography. As judged by the protein profile from the molecular sieve chromatography, the recombinant meningococcal porins eluted from the column as trimers. To be certain that no PCR artifacts had been introduced into the meningococcal porin genes to allow for such high expression, the inserted PorB gene sequence was determined. Inhibition ELISA assays were used to give further evidence that the expressed recombinant porin proteins had renatured into their natural antigenic and trimer conformation.